Superoxide dismutase (SOD) is one family of antioxidant enzyme that functions to remove damaging reactive oxygen species from cellular environment and to protect cells. SOD functions to dismutation of two superoxide radicals to hydrogen peroxide and oxygen. SOD is classified into Cu/Zn SOD containing copper and zinc atoms, Mn SOD containing manganese atom, and extracellular superoxide dismutase (EC SOD) located in the cell surface or the extracellular fluid. Of them, EC SOD contains copper and zinc atoms as in Cu/Zn SOD, but is characterized in that a heparin-binding domain is present in the C-terminal end. Since EC SOD has the heparin-binding domain, it is assumed that EC SOD will function to protect cell membranes by binding to the cell membranes. According to literatures, it was known that EC SOD plays a role in the body's defense mechanism in serums and extracellular matrices (Marklund et al, Biochem. J. 266, 213-219, 1990; Su et al., Am J Respir Cell Mol. Biol., February 16(2), 162-70, 1997; Luoma et al., Thromb. Vasc. Bio. 18, 157-167, 1998). Other literatures reported that gene therapy with EC SOD improves aorta restenosis in rabbits and alleviates collagen-induced arthritis in mice (Laukkanen M O et al., Circulation, 106, 1999-2003, 2002; Iyama S et al., Arthritis & Rheumatism, 44, 9, 2160-2167, 2001). Recently, it was reported that EC SOD could inhibit telomere shortening, a cell aging phenomenon in human fibroblasts and extends the replicative life span of human fibroblast (Serra V et al., J. Biol. Chem., 278, 9, 6824-6830, 2003). In addition, it was reported that the heparin-binding domain of EC SOD acts as a nuclear localization signal so that it is located within the nuclei of thymuses and testis cells so as to protect genomic DNA from oxidative stress and to regulate the DNA transcription sensitive to oxidation-reduction reaction (Ookawara T et al., BBRC, 296, 54-61, 2002). However, neither the distribution pattern of EC SOD in the skin nor the effect of EC SOD on skin diseases is yet known.
Meanwhile, as the fact that certain proteins can effectively enter cells through cellular membranes is found, studies to use such proteins as transport means to transduce useful substances into cells are now actively performed. Typical examples of such proteins include HIV Tat protein, ANTP, VP22 protein, PEP-1 peptide, and the like (Lindgren et al., TIPS 21:99, 2001; Green et al., Cell, 55, 1179-1188, 1988)). It is known that the cell-transduction ability of such proteins is caused by the properties of a protein transduction domain (PTD) with the activity capable of crossing a cell membrane phospholipid bilayer (Fankel A. D. et al., Cell, 55, 1189-1193, 1988; Green M. et al., Cell, 55, 1179-1188, 1988).
Accordingly, since drugs or proteins which are used as therapeutic agents cannot spontaneously cross the cell membrane in most cases, studies to use the above-described protein transduction domain to introduce useful proteins into cells are now performed.